CCR Office of Science and Technology Partnerships

1. CCR Office of Science and Technology Partnerships Administrative Support: Ms. Davida Bilgrad, bilgradd@mail.nih.gov David Goldstein, Ph.D., goldsted@mail.nih.gov Mariam Malik, Ph.D., malikm@mail.nih.gov http://ostp.nci.nih.gov October 15, 2012

2. CCR Office of Science and Technology Partnerships (OSTP) Mission • Facilitate the research of CCR researchers by ensuring access to the latest and best technologies and bioinformatics resources. • Develop partnerships, collaborations, scientific contracts, license agreements, and other technology transfer agreements with the private sector for access to hardware, software, expertise, and services. We negotiate economies of scale for significant cost benefit to CCR. • Spearhead technology-based initiatives through CCR core facilities and other NCI technology-oriented research programs. We work with these groups to make sure they have the tools they need to better serve the CCR community. • Develop web sites, databases, and training programs on relevant technologies and bioinformatics resources.

3. CCR Technologies and Scientific Resources • Protein Technologies, Resources, and Services • Antibody Development, PPI, NanPro/Simple Western • Genomics Technologies, Resources, and Services • Gene Editing Technology, NGS • Bioinformatics • Core, Software Tools, BTEP • Metabolomics Profiling Services (Metabolon and MDC) • Technology Development and Access to Core Resources • ATP and CCR core facilities • OSTP Technology Subsidy Program Areas of Focus:

4. Protein Technologies, Resources and Services • Rockland: Development of rabbit polyclonal and mouse monoclonal antibodies at no charge to investigators • Epitomics: Development of rabbit monoclonal antibodies at $5,800/antibody (70%) discount. Antibody Development CCR, through the OSTP, has established unique partnerships with companies to produce antibodies against key cancer and HIV targets of interest to CCR

5. Epitomics Partnerships for Antibody Production The development of rabbit monoclonal antibodies is highly desirable because: • Higher affinity and sensitivity • Diverse epitope recognition (post-translational modifications) • Improved immune response to mouse antigens

6. Partnerships for Antibody Production • 170 total projects - 80 participating CCR laboratories • 46 failed projects (~70% success rate) • 110 rabbit polyclonal antibodies delivered to investigators • 12 antibodies in production To request a new antibody: http://www.abcc.ncifcrf.gov/antibody_rock/atbd_login.php

7. Epitomics Partnerships for Antibody Production To request a new antibody: Contact Mariam Malim at malikm@mail.nih.gov https://epit.abcc.ncifcrf.gov/atbd_login.php • 80 total projects – 35 participating CCR laboratories • Only 9 failed projects • 50 rabbit monoclonal antibodies delivered to investigators • 20 antibodies in production

8. New Protein Interaction and Antibody Database: C-DAPI HyNet Yeast 2-Hybrid data: An extensive collection of experimentally determined and verified human protein-protein interactions (PPI). Largest human PPI database in the world, containing over 120,000 non-redundant human protein interactions covering nearly one half of the human genes in the NCBI RefSeqdatabase. CCR Database of Antibodies and Protein Interactions The Largest Y2H Data Set Now Available to all CCR investigators Access to C-DAPI: http://ostp.nci.nih.gov. Click on “Protein Technologies and Services”http://ccrdapi.abcc.ncifcrf.gov/apps/authenticate/login

9. New Protein Interaction and Antibody Database: C-DAPI

10. New Protein Interaction and Antibody Database: C-DAPI

11. New Protein Interaction and Antibody Database: C-DAPI

12. C-DAPI Bait Page

13. C-DAPI Prey Page

14. New Protein Interaction and Antibody Database: C-DAPI

15. Complex Marker Control Protein or peptide fractionation LC of peptides LPAT: PPI Discovery and Validation Novel protein complex isolation performed by affinity purification using epitope tagged proteins or primary antibody against the target. Induced expression in HEK293T or physiologically relevant cells. Target Target Affinity purification Quantitative protein complex discovery using both label free quantitation and/or SILAC. These methods can aid in the identification of transient interacting proteins and distinguishing between background proteins and specific interacting proteins.

16. DAPI Int. Merged PPI Validation using IP-Western and Imaging Western blotting: Bmi1 and RYBP interaction discovered by both affinity purification and Y2H. Validation of the interaction performed in HEK293T cells expressing increasing amount of FLAG tagged Bmi1 (upper panel) followed by co-immunprecipitation. RYBP detection correlates with amount of Bmi1 (lower panel). IP: a-FLAG-Bmi1 C S1 S2 S3 Blot: a-FLAG-Bmi1 ThorkellAndresson, Ph.D. Laboratory of Proteomics and Analytical Technologies andressont@mail.nih.gov Blot: a-RYBP Co-localization: GATA3 and Int. Control • Two different methods were used to validate the interaction between p53 and a novel interacting proteins: • Conventional confocal microscopy • O-link GATA3 O-link co-localization of GATA3 and Int. Conventional confocal co- localization of GATA3 and int.

17. Collaborative Protein Technology Resource: NanoPro System-- Capillary based isoelectric focusing Immunoassays to detect protein phosphorylation isoforms using nanogram level of protein Step 1: Load The capillary is filled with a 400-nL (~5-100ng) mixture of sample lysate, fluorescently labeled pI standards and ampholytes. Step 2: SeparateVoltage is applied across the capillary to drive the IEF separation. Individual proteins and pI standards concentrate at their isoelectric points, and the position of each standard in the capillary is recorded. Step 3: ImmobilizeThe capillary is exposed to UV light, activating the proprietary linking chemistry and locking the separated protein isoforms to the capillary wall. Step 4: ImmunoprobeThe capillary is rinsed and immunoprobed for specific proteins. Luminol and peroxide are added to generate chemiluminescent light, which is captured by a CCD camera. Step 5: QuantitateThe digital image is analyzed and quantitative results are presented in the software. • Pan reactive antibodies can be used to probe multiple protein phosphorylation isoforms • Provides quantitative and highly specific information on the activation status of signaling molecules. NanoPro

18. Simple Western System-- Capillary based automated Western system – all steps computer programmed, Gel free, blot free and hands free; – highly quantitative data with excellent reproducibility. rep. 1 rep. 2 WC NE WC NE TNFa - + - + - + - + NFkB-p65 IkB-a C-Rel IKKa Load Matrix: The capillary is filled with stacking and separation matrix Load Sample: 40nL (~5-100ng) sample containing protein lysates, fluorescent MW standards and buffers are loaded Separate & Immobilize: Proteins are separated by MW while migrating through the separation matrix, and covalently linked to the wall of the capillary through UV activated chemistry Immunoprobe: ‘Western blot’ occurs inside the capillary using a primary antibody, and a secondary antibody conjugated with HRP Quantitate: chemiluminescent signals are measured and analyzed using Compass software. Caspase 9 aTubulin Protein transfer inconsistencies are eliminated!! Low ng sensitivity!!

19. Established Capillary Immunoassays at CPTR -- CPTR, Collaborative Protein Technology Resource, a core facility specializing in developing and implementing cutting edge protein-based technologies to facilitate discovery and translational research in CCR/NCI/NIH NanoPro assays: MAP Kinase Pathway: Erk1/2: Total Erk 1/2, phospho-Erk1/2;MEK1 and MEK2 and phospho-MEK (pS218/222, pT292, pT386), AKT: pan Akt, Akt1, Akt2, Akt3, phospho-Akt (pS473, pT308) Protein Kinase C: PKCα, PKCδ, phospho-PKCδ (pS299, pY311A) JNK: phosphoJNK (pT183/pY185) STAT: STAT3 pY705, STAT5 pY694 4EBP1: (pan & pT37/46, pS65) Apoptosis proteins: Caspase 3, Caspase 8, cIAP1 Heat shock proteins: Hsp70, Hsp72, Hsp73 Epitope-tagged proteins: GFP, V5, Flag, etc Loading Controls: β2-microglobulin, ALAS1, thioredoxin 1 Other NanoPro assays: Visit the ProteinSimple website at www.proteinsimple.com (select literature then NanoPro) Samples tested in NanoPro system: Cultured mammalian cells: breast, prostate, pancreatic, ovarian, liver cancer cell lines, and Stem cells etc. Tissue samples: Xenograph, mouse lung, mouse skin etc. Patient biopsies: peripheral blood mononuclear cells (PMBCs), CD138+ multiple myeloma cells, solid tumor fine needle aspirate etc. Simple Western Assays: MAP Kinase Pathway: Erk1/2, MEK1, MEK2 AKT: Akt (pan), phospho-Akt pS473 AMPK (energy homeostasis): AMPKα, AMPKα pS485 mRNA Translation: P70 S6 Kinase, P70 S6 Kinase pT389 , S6 ribosomal protein, S6 ribosomal protein pS235/236 Apoptosis: c-IAP1 ,Caspase 3, Cleaved Caspase 7, Parp, Cleaved Parp, p53 Protein Kinase C: PKCδ, phospho-PKCδ pS299,PKCβ , PKCε, PKCµ, PKD1 pS744/748 Membrane receptors: EGFR NFκB: c-Rel, I-κκ-B-α, IκBα pS32/36, I-κκ-α, NF-κ-B p65 , NF-κ-B1 p105/p50 , NF-kappa-B2 p100/p52 Cell cycle: MCM2, MCM4, MCM5, KID, SMARCB1, TACC3 Transcription regulators: STAT3 ,STAT5, c-Myc, SMAD1,2,3,and 5 RASGRP3: RasGRP3, RasGRP3 pT133 TNF receptor-associated factors:Traf1 Epitope-tagged proteins: GST, etc Loading controls: α-tubulin ,β2-microglobulin, GAPDH, β-actin In development: 4E-BP1, AMPKβ, c-IAP2, RIP, FLIP Contact Information Jinqiu (Jessie) Chen, PhD (Program Manager) chenj13@mail.nih.gov, 301-496-7517; Michelle Herrmann, MS herrmannma@mail.nih.gov, 301-594-3749 Location: Bldg. 37, Rm. 2140, Bethesda Campus Developing new capillary Immunoassays based upon the demands of the CCR community

20. New Partnership for Gene Editing Highly-efficient and proprietary gene-engineering platform technology called GENESIS™ GENESIS permits routine alteration of any endogenous DNA sequence in human cells to create isogenic lines. The technology is based on rAAV (recombinant adeno-associated virus) mediated homologous recombination: First technology that enables precise engineering of any DNA variation in any human cell line, including point mutations and SNPs.

21. Examples of what you can do with rAAV G12V Knock-ins/point mutations Constitutive gene knock-outs Inducible gene knock-outs KI Tags/cDNA’s/reporters

22. Partnership for gene engineering of cell lines through the ATP in Frederick •  ATP labs now provides NCI investigators with the ability to modify genes in vivo in cell lines using Horizon’s rAAV-mediated gene replacement technology.  • Vector Engineering for knock-in epitope tags, SNPs, or other genetic alterations on various genes. (Cloning Optimization Group) • Generation of AAV viruses (PEL Virus Optimization Group) • Generation of mutant cell lines (PEL Eukaryotic Expression Group) • Tandem affinity purification to identify protein complexes (LPAT Protein Functional Characterization Group) Currently 4 labs working with Horizon and the ATP

23. CCR Sequencing Facility (SF) Providing high-throughput sequencing technologies to identify single nucleotide polymorphisms, insertions and deletions, and copy number or structural variations Director: Mr. Bao Tran: tranb2@mail.nih.gov • Access to the latest NGS technologies. Consultation throughout the design and execution of sequencing projects. • Platforms: Illumina Genome Analyzer IIx, HiSeq 2000, and PacBio platforms. • Sequencing applications cover a wide range of experimental protocols and can be adjusted to fit the specific needs of your laboratory and budget. • Service and Collaboration: Facility staff provide consultation throughout the experimental design and execution process, including preparation of sequencing requests, sample submission and tracking, and delivery of sequence data. http://atp.ncifcrf.gov/genetics-and-genomics/sequencing-facility/

24. CCRIFX Bioinformatics Core • Main Points • Shared effort between CCR and SAIC-Frederick • Mission: To enable CCR researchers from both Bethesda and Frederick campuses with the management and analysis of the massive amounts of data emerging out of experiments relating to high-throughput advanced biomedical technologies, such as microarrays, proteomics, and next-generation sequencing  • Primary Expertise Areas • ChIPseq: Mapping of DNA binding proteins across the genome • RNAseq: expression profiling, SNP calling/Variant Analysis, etc. • Microarray: mRNA/miRNA expression profiling • Pathway mapping and biological interpretation • Cost • None…

25. CCRIFX Website http://ccrifx.cancer.gov

26. CCR/OSTP Bioinformatics Toolbox • Genomatix Suite: • Extended genome annotation/promoter mapping • BiblioSphere: Literature mining and pathway mapping • ChipInspector: Microarray analysis • NEW!! The Genomatix Genome Analyzer: an integrated solution for comprehensive second-level analysis of Next Generation Sequencing (NGS) data from ChIP-Seq, RNA-Seq or genotyping experiments. • Pathway/Network Analysis Tools: • Ingenuity Pathway Analysis (IPA) • Elsevier Ariadne Pathway Studio • GeneGo MetaCore (now with Thomson/Reuters)

27. CCR/OSTP Bioinformatics Toolbox • Partek: Genomics Suite: Statistical analysis and visualization for microarray and Next Generation sequence data • GeneSpring customized license that integrates: • Microarray analysis • arrayCGH analytics • Ingenuity IPA integration • BioDiscovery: Nexus arrayCGH analytics and Gene Expression Analysis • CLC bio: Analysis and visualization of next generation sequencing data • NEW!! Geneious Pro: Geneious is a DNA, RNA and protein sequence alignment, assembly and analysis software platform

28. Bioinformatics Training and Education Program http://bioinformatics.nci.nih.gov/training/

29. OSTP Technology Subsidy Program • Subsidy available: • 100% for piloting of emerging technologies • 50% up to $10,000 total subsidy/PI for new technologies • 33% up to $10,000 for standard genomic technologies • Projects above $20,000 total cost should be submitted to RRS • Microarray Platforms including full service analysis (through ATP) • NanoString digital gene expression profiling • Protein Detection and Interaction Screening (through ATP) • Antibody Development: Epitomics and Abpro • NanoPro/Simple Western capillaries • MetabolonScreening • Whole genome sequencing and specialized Next Gen. sequencing projects Supported technologies: http://ostp.nci.nih.gov: Click on “OSTP Technology Subsidy Program”

30. Additional funding source for the development and application of new technologies NCI-Frederick Office of Director Research Support (ODRS) managed by the OSTP • 50% Funding for Collaborative Projects with the Advanced Technology Program (ATP) • Not readily categorized as Technology Development or Core Service projects • Represent the transition of a Technology Development project into a new research area • Represent feasibility or pilot studies which will serve as a starting point for future Core Service projects • Focus the direction and provide validation of Technology Development

31. CCR Office of Science and Technology Partnerships Administrative Support: Ms. Davida Bilgrad, bilgradd@mail.nih.gov David Goldstein, Ph.D., goldsted@mail.nih.gov Mariam Malik, Ph.D., malikm@mail.nih.gov http://ostp.nci.nih.gov October 15, 2012

32. CCRIFX Bioinformatics Core • Getting started • Submit request at http://ccrifx.cancer.gov. (Use NIH NED login id and password to access.) • Required request information: PI, lab name, data availability date, desired completion date, name for request, basic description of support needed • Follow-up • CCRIFX staff contact you to gather more information • CCRIFX helps characterize and deliver requested analysis • If request is out of core expertise areas, others may be asked to assist • Suggestions • Contact CCRIFX early, in the experimental design phase • Use office hours to get to know core capabilities • Many more details available at CCRIFX website including FAQ!

33. BTEP Training and Lecture Schedule

34. Examples of OSTP Technologies and Partnerships • Robust technologies for Antibody Development • Highly-efficient and proprietary gene-engineering platform technology: Horizon Discovery • Digital Gene Expression Profiling: NanoString • Next Generation Sequencing Technologies: Illumina, Complete Genomics, Knome • Database of Protein-Protein Interactions (Yeast 2-Hybrid): C-DAPI • Tool to identify causal variants from human resequencing (NGS) data: Ingenuity • Tools for analysis of DNA sequencing data: CLCbio, Geneious Pro, Genomatix GGA/GMS